Background:
Lynch syndrome (also known as Hereditary Non-Polyposis Colon Cancer, HNPCC), is an autosomal dominant hereditary cancer syndrome that accounts for 3-5% of all colon cancers. Lynch syndrome-associated tumors are characterized by microsatellite instability (MSI) and are caused by germline mutations in any of four mismatch repair (MMR) genes (MLH1, MSH6, MSH2, PMS2). The risk of colon and gastric cancers are increased in both sexes, and women with Lynch syndrome have an increased risk for endometrial and ovarian cancers.
The testing strategy for Lynch syndrome includes screening by MSI analysis followed by immunohistochemistry (IHC) testing of MMR proteins. Full gene sequencing can then be performed to identify germline mutations in the putative mutated gene(s) identified by IHC (please contact our lab for more information regarding MSI and IHC testing). Germline mutations in MLH1 and MSH2 account for 90% of Lynch syndrome cases while mutations of MSH6 and PMS2 comprise the remaining fractions. While one study indicated that 65% of MSH2 and 87% of MLH1 variants were point mutations (the rest being gross deletions/duplications), comprehensive clinical sensitivity of MSH2, MLH1, and MSH6 sequencing is unknown.
Reasons for Referral:
- Unknown familial mutation in MMR gene(s) with a positive family history of colorectal cancer without IHC and/or MSI information
- Identification of inherited genetic defects in MMR gene(s) in colorectal cancer patients with tumors testing positive by IHC and/or MSI
- Predispositional testing for asymptomatic family members with a positive family history of colorectal cancer
Methodology:
Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs <50 bp, the analytical sensitivity is >87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.
Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmental duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.
This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.
Specimen Requirements:
Blood: EDTA or ACD (Solution A or B):
- Adult: 5mL
- Child: 5mL
- Infant: 2-3mL
Saliva: 2 ORAgene™ Saliva Collection Kits (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Assisted Saliva: 4 ORAgene™ Assisted Saliva Collection Kits (OGR-575) used according to manufacturer instructions. Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.
Buccal Cells: 4 CytoSoft™ Cytology Brush (Medical Packaging CYB-1) used according to manufacturer instructions. Please contact KDL Client Services for a Buccal Collection Kit for patients that cannot provide a blood sample.
Skin Fibroblast: Punch Biopsy (Cell cultures will be prepared at KDL and used for testing), or 2 T-25 confluent flasks
DNA: 5-10µg at a minimum of 100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS).
For routine testing of blood and saliva (or DNA extracted from these specimens), KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed within the above windows, please contact the laboratory.
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including ethnicity, clinical history, and family history.
Test Performed (Days):
Weekly
Turn Around Time:
6-8 weeks
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold, but not frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
References:
- Balmaña J, Stockwell DH, Steyerberg EW, et al. Prediction of MLH1 and MSH2 Mutations in Lynch Syndrome. JAMA: The Journal of the American Medical Association. 2006;296(12):1469 -1478.
- Bonadona, V. et al., 2011. Cancer Risks Associated With Germline Mutations in MLH1, MSH2, and MSH6 Genes in Lynch Syndrome. JAMA: The Journal of the American Medical Association, 305(22), pp.2304-2310.
- Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group, 2009. Recommendations from the EGAPP Working Group: genetic testing strategies in newly diagnosed individuals with colorectal cancer aimed at reducing morbidity and mortality from Lynch syndrome in relatives. Genetics in Medicine, 11(1), pp.35-41.
- Goodfellow, P.J. et al., 2003. Prevalence of defective DNA mismatch repair and MSH6 mutation in an unselected series of endometrial cancers. Proceedings of the National Academy of Sciences, 100(10), pp.5908-5913.
Additional Info: